The development of fully effective vaccines against many pathogens would be of enormous benefit to society in reducing morbidity, mortality, and health care costs. Unfortunately, current vaccines often generate suboptimal immune responses. The overall theme of this application is to manipulate the underlying mechanisms of antigen presentation to enhance the immunogenicity of vaccines. There are 3 specific Aims: 1. Engineering antigens in recombinant DNA-based vaccines for enhanced presentation through the endogenous MHC class I pathway. The hypothesis underlying this Aim is that antigen processing is a key rate limiting step in the presentation of antigens with MHC class I molecules. The goal of this set of experiments is to develop methodology to enhance the processing of antigens and thereby generate more peptide-MHC complexes and stronger T cell responses. Our experimental approach will be to develop generic modifications of genes encoding antigens that target the modified gene products for processing by the endogenous class I pathway. This approach is based on new insights into the class I processing pathway. These constructs will then be tested sequentially for enhancing immunogenicity in vitro, in vivo and ultimately for their efficacy in vaccines in specific disease models. 2. Engineering antigens in recombinant DNA-based vaccines for enhanced presentation by the MHC class II pathway. The hypothesis and goals of this aim are similar to Aim 1 but are directed at enhancing the presentation of antigens with MHC class II rather than class I molecules. Our experimental approach will be to develop modifications of genes encoding antigens that will target the antigens into appropriate subcellular compartments for them to efficiently enter the MHC class II pathway. This is a particularly important approach for developing effective DNA-based vaccines because most endogenously synthesized antigens do not efficiently gain access to the class II pathway in cells. These constructs will then be tested for enhancing immunogenicity in vitro and in vivo and for their efficacy in specific disease models. 3. Concomitant administration of cytokine and costimulatory genes to enhance T cell priming to recombinant DNA-based vaccines. The rationale for this Aim is that the key adhesion molecules and costimulatory factors that are necessary for optimal antigen presentation and T cell responses are known. The expression of many of these factors is regulated by cytokines and cytokines also stimulate antigen presentation pathways. The hypothesis underlying this Aim is that immunogenicity can be enhanced by upregulating adhesion molecules and costimulatory factors as well as the generation of peptide-MHC complexes at the site of antigen presentation. The experimental approach will be to develop DNA-based vaccines that locally co-express cytokine and/or costimulatory genes together with the antigens developed in Aims 1 and 2. These constructs will then be tested for enhancing immunogenicity and efficacy in vivo.